The 31.2% decrease in HIV acquisition reported in the RV144 trial has raised hopes that vaccine protection may be achievable. We have pursued a bimodal vaccine approach to induce both cellular and humoral immunity; in our recent rhesus macaque (RM) study, recombinant protein immunogens (SIV Gag-Pol particles, HIV Tat, and multimeric HIV clade C (HIV-C) gp160) provided complete protection for some RMs against multiple intrarectal challenges with a heterologous R5 clade C SHIV (SHIV-C). Our study simultaneously linked cellular as well as humoral antiviral immunity to protection. Overall, five vaccine-protected RMs remained free of persistent, systemic infection; all had generated high-titer neutralizing antibodies (nAbs) in response to multimeric gp160 of an HIV-C strain that diverged by 22.2% in amino acid sequence from Env of the challenge virus. Our overall hypothesis is that vaccine-induced Abs - either nAbs and/or Abs with antiviral effector functions - can protect against heterologous virus acquisition. We have developed new tools to determine the epitope specificity of protective Abs from polyclonal sera. Our strategy involves a) differentil biopanning with recombinant peptide libraries to isolate mimotopes linked to protection, b) isolation of single RM B cells specific for a given mimotope/epitope, 3) PCR amplification of RM immunoglobulin variable regions, and 4) generation of recombinant Abs. These novel approaches have led to the isolation of two new chimeric simian/human nmAbs with predicted epitope specificity. We now seek to use these new tools for the following Specific Aims: 1. to characterize the epitopes recognized by polyclonal Abs of vaccine-protected RMs by differential biopanning. First, we will positively select recombinant phages encoding random peptide libraries by biopanning with plasma from a vaccine-protected RM, followed by negative counter-selection with plasma from vaccinated, unprotected RMs. After several rounds of positive/negative selection, recombinant phages will reflect mimotopes linked to protection. We will also address the converse question: did RMs with vaccine failure mount unfavorable Ab responses that are not found in vaccine-protected RMs - or is failure simply a lack of protection-linked Abs? To do this, we will reverse the biopanning strategy and characterize the cognate epitopes linked to vaccine failure. 2. to isolate antigen-specific single B cells from the protecte RMs and PCR amplify the heavy/light chain variable immunoglobulin regions, using our newly generated RM-specific primers. 3. to perform passive immunization in RMs with the novel mAbs to demonstrate protection against mucosal challenge with a heterologous R5 SHIV. Our studies, which are based upon a well-characterized cohort of vaccine-protected RMs given upfront heterologous SHIV-C challenges, will identify epitopes that are protective or perhaps also deleterious in vivo and therefore provide important information for future HIV/AIDS immunogen design and optimization.